Principal Supervisor: Dr. Allister Crow, Department of Pathology, University of Cambridge
Co-supervisor: Professor David Roper, School of Life Sciences
PhD project title: Structural insight into the role of FtsEX in bacterial cell division
University of Registration: University of Warwick
FtsEX is an ABC transporter-like protein complex composed of a cytoplasmic ATP-Binding Cassette (FtsE) and multi-pass integral membrane protein (FtsX) with a large periplasmic domain located between its first and second transmembrane helices.
During cell division, FtsEX coordinates the activities of the cytoplasmic cell division machinery with those of the periplasmic peptidoglycan hydrolases responsible for breakdown of the cell wall. E. coli FtsEX interacts directly with tubulin-like FtsZ in the cytoplasm and periplasmic adaptors such as EnvC and NlpD in the periplasm. EnvC and NlpD subsequently interact with, and activate, peptidoglycan hydrolases (e.g. AmiA, AmiB and AmiC) leading to localised enzymatic degradation of peptidoglycan at the site of cellular constriction.
It has been hypothesised that FtsEX may undergo long-range conformational changes that serve as a transmembrane signal between binding partners on either side of the bacterial membrane to synchronize the division process, but the nature of these conformational changes, and the role of ATP hydrolysis in this process, remain poorly understood.
This project will address the structure and mechanism of FtsEX using a combination of X-ray crystallography and other biophysical tools. The project will address four key questions: Firstly, how does FtsEX bind and activate periplasmic proteins associated with breakdown of peptidoglycan? Secondly, what is the role for ATP binding and hydrolysis by FtsEX during cell division? Thirdly, how does FtsEX interact with the cytoplasmic components of the cell division machinery? Finally, do conformational changes in FtsEX mediate ‘transmembrane signaling’ between the cytoplasmic and periplasmic components of the divisome? If so, what do these structural changes look like?
To understand how FtsEX activates periplasmic peptidoglycan hydrolases, we will characterise the interaction between a soluble extracytoplasmic domain of FtsX and its periplasmic substrates (such as EnvC) using isothermal titration calorimetry (ITC) and/or size exclusion chromatography (SEC). We will also co-crystallise the FtsX periplasmic domain in complex with its interaction partners to reveal details of the molecular interface.
To investigate the role of ATP, we will measure the ATPase activity of FtsEX and test whether addition of cytoplasmic or periplasmic binding partners (e.g. FtsZ or EnvC/NlpD, respectively) stimulate or repress ATPase activity of FtsEX activity. Using non-hydrolysable ATP analogues, we will further dissect how the nucleotide status of FtsEX affects its ability to interact with cytoplasmic and periplasmic binding partners.
To characterise interactions between FtsEX and its cytoplasmic binding partners we will make quantitative measurements of the affinity between FtsEX and FtsZ using ITC and map the interaction interface using site-directed mutagenesis approach.
To elucidate long-range conformational changes in FtsEX that might mediate transmembrane signalling, we will determine its structure using X-ray crystallography. To maximise the chances of successful crystallisation, we will screen multiple bacterial homologues of FtsEX in a range of detergents, both with and without nucleotides. A structure of FtsEX presents the best opportunity to identify protein features capable of directing transmembrane conformational change and, ultimately, to understand how FtsEX works.
In summary, a complete structural and biophysical characterisation of FtsEX and its interaction partners on either side of the bacterial inner membrane will provide deep molecular insight into how cell division is regulated, and open up new opportunities to use this knowledge to disrupt, delay and inhibit the process through development of new antibacterial agents.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
Purification of both soluble and membrane proteins.
Use of X-ray crystallography to determine macromolecular structures.
Development of structure-based hypotheses of protein mechanism and testing of these ideas using in vitro and in vivo techniques.
Additional methods may include simple DNA work such as cloning genes for expression in bacteria and site-directed mutagenesis to dissect structure-function relationships. Isothermal titration calorimetry will be used to characterise protein:protein interactions.
Contact:Dr. Allister Crow, Department of Pathology, University of Cambridge